Fibronectins are disulfide-bonded dimers found in vertebrates (e.g. mammals, birds, amphibians, fish, reptiles) that have been implicated in cell adhesion, wound healing, and embryogenesis. See E. Ruoslahti, 57 Ann. Rev. Biochem. 375-413 (1988); J. Thiery et al., in Fibronectin181-212 (Academic Press 1989); R. Colvin, in Fibronectin213-254 (Academic Press 1989); K. Yamada, in Fibronectin47-121 (Academic Press 1989).
The majority of fibronectin consists of three types of repeating homology units. T. Petersen, et al., 80 P.N.A.S. U.S.A. 137-141 (1983); T. Petersen et al., in Fibronectin163-179 (Academic Press 1989). Type I repeats contain two intrachain disulfide bonds and are present in the amino and carboxy-terminal regions of the molecule. Type II repeats are found only in a gelatin binding region of fibronectin and also contain two intrachain disulfide bonds. There are also 15-17 copies of type III repeats, which are located in the central portion of the molecule and lack intramolecular disulfide bonds.
Recent studies have shown that fibronectin becomes insolubilized into fibrils following binding to specific sites on the cell surface, termed matrix assembly sites. This binding is mediated by an amino-terminal 70 kDa fragment of fibronectin. The 70 kDa fragment contains nine copies of the type I repeat and two copies of the type II repeat. While much is known about the structure of fibronectins, the possibility of using all or part of a gene coding for the fibronectin gelatin binding region in a purification vector has not previously been suggested.
In connection with recombinant DNA technology it is often desirable to amplify large quantities of a vector (e.g. plasmid; phage), express proteinaceous material in a cell using the vector, cause the proteinaceous material to be secreted from the production cell into the surrounding media, and then purify the protein from the surrounding media. However, this process had not previously been optimized for expression of certain proteins. For example, while vectors had been created that efficiently express proteins, these proteins were often folded incorrectly, secreted inefficiently, or were difficult to separate from the surrounding media once secreted. These deficiencies caused reduced yield, impurities in the final product, and required the use of overly expensive and time consuming purification techniques.
A particularly desirable feature for techniques for expressing and purifying proteins via recombinant techniques is the ability to create a final product that does not contain extraneous fusion amino acids. This is especially critical in reducing regulatory delays in obtaining approval for use of such proteins.